Chronic viral infections remain major threats to global health, with pathogens such as hepatitis B virus (HBV) and hepatitis C virus (HCV), responsible for millions of deaths annually. The recent development of curative antiviral therapy for HCV has been a major breakthrough. However, therapies capable of producing at least functional cure for chronic HBV are urgently needed. A major impediment to cure of HBV is a functionally impaired immune response characterized by markers of exhaustion. The PD-1:PD-L1/2 inhibitory receptor pathway regulates many key aspects of cellular immunity, including T cell exhaustion in chronic viral infection and cancer. Blockade of this pathway has produced dramatic effects in the treatment of advanced cancer and unleashed an immunotherapeutic revolution. However, little is known about the human in vivo effect of PD-1 blockade on the response to chronic infections marked by exhaustion and the mechanisms by which these responses are invigorated. Building on our U19 CCHI Consortium?s important work defining the mechanisms of immune exhaustion in chronic HCV and assessing its reversal upon termination of chronic antigen stimulation, we will now comprehensively investigate how direct and specific blockade of PD-1, as a key mediator of antigen- mediated immune exhaustion, affects the layers of molecular regulation of the antiviral immune response. The overall hypothesis of this project is that blocking PD-1 in humans will alter the magnitude, quality, regulation and composition of pre-existing antiviral CD8 T cell responses, leading to more effective viral control, and will modulate other aspects of cellular immunity, including atypical T cell and macrophage responses. We will test this hypothesis through the following aims. Specifically, in Aim 1 we will test how PD-1 therapy alters pre- existing virus-specific CD8 T cell responses targeting persisting viruses in the blood. We will utilize large PBMC donations from leukapheresis for a comprehensive and in-depth analysis of changes in the phenotype, function, clonal composition, transcriptional state, and epigenetic regulation of HBV-, but also CMV-, EBV-, HBV- and influenza-specific CD8 T cells, as well as of unconventional MAIT T cells. In Aim 2 we will test how PD-1 therapy alters HBV-specific CD8 T cell responses in the liver of patients with chronic hepatitis B. We will complement our analyses from aim 1 with parallel data directly from the site of infection through liver fine needle aspirates. Finally, in Aim 3 we will define the recovery of macrophages through PD-1 blockade in persons with chronic HBV infection. These data will extend our analysis on the effects of PD-1 blockade to other components of the antiviral immune response, by studying the response of macrophages as critical modulators of intrahepatic innate immunity. Collectively, we expect these data to dramatically enhance our understanding of the mechanism of ?PD-1 mediated immune recovery that can be utilized not only for the design of pathogen- specific immunotherapy, but also to enable further improvements in the efficacy of immunotherapy in general.